N-Guanidylacetyl-{8 des-asp{hu 1{b -Ile{hu 8{b {9 {0 angiotensin II as an angiotensin antagonist

ABSTRACT

A new octapeptide having profound angiotensin antagonist properties has been found. The new compound resembles angiotensin II except for the two terminal aminoacids, carrying an unnatural aminoacid at the N-terminus.

United States Patent I19] Bumpus et al.

1 1 Dec. 2, 1975 1 N-GUANIDYLACETYL-[DES-ASP'JLEH ANGIOTENSIN 11 AS AN ANGIOTENSIN ANTAGONIST [22] Filed: May 10, 1974 [2]] Appl. No: 468,854

[52] U.S. Cl. 260/112.5 R; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/00 [58] Field of Search 260/112.5

[56] References Cited UNITED STATES PATENTS 3,8l6,385 6/1974 Gillesser et al. 260/l 12.5

Primary ExaminerLewis Gotts Assistant ExaminerReginald J. Suyat Attorney, Agent, or FirmPaul D. Burgauer; Robert L. Niblack 4 [57] ABSTRACT A new octapeptide having profound angiotensin antagonist properties has been found. The new compound resembles angiotensin ll except for the two terminal aminoacids, carrying an unnatural aminoacid at the N-terminus.

1 Claim, N0 Drawings N-GUANIDYLACETYL-lDES-ASP ELE I ANGIOTENSIN II AS AN ANGIIOTENSIN ANTAGONIST DETAILED DESCRIPTION OF THE INVENTION This invention relates to polypeptides. More particu larly, it is concerned with the octapeptide of the formula:

which is more easily referred to as the octapeptide chain identified as:

C-uaAc-L-Arg-L-Val-L-Tyr-L-lle-LJ-lis-bPro-L-lle (2) The peptide of this invention possesses valuable pharmacological activity. It is capable of inhibiting the pressor effect of angiotensin II. Thus, when administered by intravenous infusion into rats in a very small amount, the pressor effect of angiotensin II is inhibited. By virtue of this inhibitory property upon angiotensin II induced blood pressure elevation, the peptide of this invention is a valuable agent for counteracting hypertension due to angiotensin II. It is also capable of reducing blood pressure in acute unilateral renal hypertensive rats upon intravenous infusion.

The octapeptide of this invention is readily prepared in accordance with known methods for preparing peptides. Such methods involve the building of a linear chain of aminoacids through repetitive amide linkages employing in such sequential alignment the necessary protective groups susceptible of ready removal by conventional cleavage methods which do not affect the peptide bonds. The adaptation of such methods to the peptide of this invention is described in the example below. All the aminoacids used in the above structure of Formula I are in the L-configuration. The correspond ing chain with D-aminoacids does not show the pharmacological effect described below.

In order to illustrate the method for making the compound of structure I, reference is made to the following example, which however, is not intended to limit the invention in any respect. In this example, the abbreviation BOC- is used in its accepted meaning, referring to tertiary butyloxycarbonyl. It also should be understood that all aminoacids used in this example are in their L-configuration except the guanidylacetic acid.

2 The BOC- aminoacids used below are either commercially available or were prepared according to the method of Schwyzer, et al., Helv. Chim Acta., 42, 2622 1959).

EXAMPLE A solution of 4.6 g of BOC-isoleucine and 2.8 ml. of triethylamine in 25 ml. of ethanol was added to 20 g. of chloromethylpolystyrene/divinylbenzene (98:2) copolymer of a mesh size between 200 and 400, containing 5.02% chlorine. The mixture was stirred at C. for 36 hours. The esterified polymer was filtered, washed several times, in sequence, with ethanol, dilute acetic acid, water, ethanol and methanol. The polymer was dried in vacuo over phosphorous pentoxide. Hydrolysis of an aliquot of the polymer and subsequent aminoacid analysis indicated that 0.4 millimoles of BOC-isoleucine were esterified per gram of the polymer. Further coupling of the BOC-proline, BOC-N- irnidazole-benzyl-histidine, BOC-isoleucine, BOC-(O- benzyl)- tyrosine, BOC-valine and BOC-nitroarginine in the respective order was carried out by utilizing the action given below for each aminoacid residue. Unless specified, all washings were carried out three times for three minutes each, first with glacial acetic acid and second with methylene chloride. The BOC group was removed by treatment with 40% (volume/volume) of trifluoroacetic acid in methylene chloride for 30 minutes, preceded by a prewash with this reagent for 3 minutes to avoid dilution of the trifluoroacetic acid by the previous methylene chloride wash. The deprotected aminoacid polymer ester was washed five times for 3 minutes each with chloroform and the trifluoroacetate salt neutralized by treating the residue for 7 minutes with 10% triethylamine in chloroform, followed by three 3-minute washings with chloroform and methylene chloride in sequence. The subsequent incoming BOC-aminoacid was added in a 2-fold excess in methylene chloride and the mixture was stirred for 10 minutes. In the case of BOC-nitroarginine and BOC- benzyl histidine, these materials were first dissolved in dimethylforrnamide, followed by filtration and mixing the filtrate with one-third volume of methylene chloride. Both of these derivatives were used in 3 -fold excesses. Coupling was aided in each instance by the addition of a 2-fold excess of dicyclohexylcarbodiimide in methylene chloride and mixing was carried out for 2.5 hours except in the coupling steps involving Arg or His where DCI was used in a 3-fold excess and mixing allowed for 8 hours. The polymer-peptide chain was then washed with DMF followed by a washing with DMF/methylene chloride (1:1) and the coupling step with the BOC-aminoacid and DCI was repeated using a mixture of 1:1 DMF/methylene chloride as the solvent. The polymer chain was then washed with methanol to remove dicyclohexylurea and finally washed with DMF. Completeness of each coupling at intermediate stages was checked by known color reaction tests. The apparatus used for the above synthesis was of the manual type described by Khosla, Smeby and Bumpus, Science, 156, 253 (1967). All couplings were carried out at C. to avoid racemization; l-hydroxybenzotriazole was used as an additive to minimize racemization of histidine [see G. C. Windridge and E. C. Jorgensen, J.A.C.S., 93, 6318 (1971)]; during the coupling of BOC-imidazolebenzyl histidine.

In the above sequence, the blocked (protected) amino-acids were coupled in sequence to the isoleucine-polymer, using BOC-proline, BOC-N-imidazolebenzyl-histidine, BOC-isoleucine, BOC-(O-benzyl)- tyrosine, BOC-valine and BOC-nitroarginine to produce a peptide of the structure of formula I with aminoacids 2 8 bound to the polymer substrate.

Guanidine acetic acid (GuaAc) was protected by adding g. of GuaAc in portions to a mixture of 40 ml. of fuming sulfuric acid and 40 ml. of fuming nitric acid at l0 C. over a period of 1 hour with vigorous stirring which was continued for another 3 hours, producing a clear, colorless solution. This solution was made alkaline in known fashion with ice and ammonia at 0 5 C. Any lumps were powdered and the mixture was acidified again with aqueous hydrochloric acid, allowed to stand at 5 C. for 10 minutes and filtered. After washing 5 times with cold water and 5 times with N-hydrochloric acid, the residue was crystallized from water to give 8 g. of nitro-Gua'Ac, melting at 186 -7.

The new compound was coupled to 4 g. of the above heptapeptide resin ester by the general procedure described above except that a 1:1 mixture of DMF and methylene chloride was used as the coupling medium. The coupling step was repeated three times to ensure completion.

The protected peptide polymer was suspended in approximately 100 ml. of freshly distilled trifluoroacetic acid and a slow stream of hydrogen bromide, prewashed with 10% resorcinol in acetic acid, was passed through the suspension under anhydrous conditions for about 30 minutes with occasional shaking. The suspension was filtered and the polymer was washed with trifluoroacetic acid. The combined filtrates were evaporated at room temperature in vacuo. The amorphous powder was washed with ether, dissolved in a mixture of methanol/acetic acid/water 101111 and the solution hydrogenated at 3.5 kg./cm. over 0.5 parts of palladium black per part of peptide weight for 48 hours with shaking. The product was purified on a 5 X 80 cm. column of Sephadex G 25 (a partially cross-linked dextran gel having an exclusion of molecular weight sizes of 5000, marketed by Pharmacia of Uppsala, Sweden) using n-butanol/pyridine/water (10:2:5) as the developing solvent. The average yield of the product obtained in this manner varied between 40 and 60% based on the millimoles of C-terminal amino acid esterified onto the polymer. Fractions in column chromatogra- 4 phy were out without regard for yield to obtain the desired compound in the pure form and no attempt was made to rechromatograph the minor fractions for identification purposes. The homogeniety of the compound was determined by thin-layer chromatography in various solvent systems of different pH, electrophoresis at pH 1.95 and 8.6 and amino acid analysis, proving that the comound is homogenous with R; 0.57 (n-butanol/acetic acid/water 4:125) and R 0.75 (n-butanol/ethylacetate/acetic acid/water 1112121), R; 0.05 (nbutanol/pyridine/water 10:2:5), R, 0.78 (n-butanol/acetic acid/water/pyridine 15:3: 1 2:10) on cellulose thinlayer plates. The chemical analysis showed that the required amino acids were present in the expected ratio.

The above antagonist was studied both in vitro and in vivo. The in vitro assay mainly evolves around an assay of isolated rabbit aortic strips mounted in a muscle bath in 5 ml. of Krebs solution.

The strips were placed under 2 g. of passive tension and allowed to equilibrate for 1.5 2 hours. Cumulative dose-response curves were obtained for angiotensin 11 at concentrations of 1 to 64 ng./ml. The antagonist was added to the bath at a certain concentration, allowed to equilibrate for 5 minutes and the response to angiotensin 11 again tested. The various doses of the antagonist thus tested were 1, 10, and 1000 ng./ml. Changes in these curves were plotted and measurement of shift to the right of the dose-response curve were measuredv At the same time, it was assured that the maximal response of the tissue remained unchanged. At the end of the experimental period, the antagonist was washed out and cumulative dose-response curves were repeated again. Results of these studies are expressed in the form of pA values as defined by 0. Arunlakshna and H. O. Schild, Brit. J. Pharmacol, 14, 48 (1959). The compound of formula I showed a pA value of 9.21 i 0.15.

In the in vivo assays, rats were anesthetized with sodium amytal and further treated with 0.6 mg. atropine and pentolinium tartrate. They were vagotomized and direct recording of the blood pressure was measured through the carotid artery, while the femoral artery was used for infusing the deisred dose over a period of 30 minutes in a physiological saline solution.

The dose-response curves of angiotensin II at 0.9 to 54 ng./kg./min. were compared by linear regression analysis with those of the antagonist at doses of 1, 10, 100 and 11000 ng./kg./min. Infusion of the above antagonist into rats thus prepared led to the initial transient pressor response of 0.7 on a scale set artificially at 100 angiotensin 11 using the same system.

For determining its antagonistic properties the compound was infused into rats at a dose-level of 250,500, 1250 ng./kg./min. The blocking effect of the compound was calculated from the dose-response curve angiotensin 11 before and during the infusion of the analog. General procedure adopted for this assay has been described by Bumpus et al., Circ. Res. 32-33 (Supplement 1), l (1973). The compound blocked the pressor response of angiotensin II at a dose level of 250 ng./kg./min. or greater.

Since the above compound is particularly suitable for injection or infusion, it is particularly valuable that the compound is water-soluble. A suitable dosage unit can be prepared by simply dissolving the above compound in water or physiological saline at a concentration of between 50 and 6000 ng./m1. Such a solution can be administered directly or it can be stored under proper The duration of action of the compound can be prolonged by injecting an oil solution intramuscularly. Oils suitable for this purpose are cod liver oil, sesame oil, refined coconut oil, etc. Antagonistic effects are thus observed for 24 hours or longer after a single i.m. injection of the drug dissolved in such an oil.

What is claimed is:

l. The octapeptide quanidylacetyl-L-Arg-L-Val-L- l Tyr-L-Ile-L-His-L-Pro-L-Ile. 

1. THE OCTAPEPTIDE QUANIDYLACETYL-L-ARG-L-VAL-L-TRY-LILE-L-HIS-L-PRO-L-ILE. 